HIV-1 infection of the central nervous system (CNS) occurs in a majority of AIDS patients, and cause neuron cell death and neurological dysfunction. The magnitude of neuronal dysfunction appears disproportionately large compared to the small number of HIV-1 infected macrophages/microglia, suggesting that indirect mechanisms are involved. Microglia/macrophages are the major target cells for HIV-1 infection in the CNS. Astrocytes occupy a large portion of the cerebral cortex, and are also susceptible to HIV-1 infection but to a restricted degree. Astrocyte apoptosis is induced by HIV-1 infection both in vitro and in vivo. While HIV-1 infection and pathogenesis is not yet understood in many systems, the process of HIV-1 infection of astrocytes is largely unknown. Our long-term objective is to identify mechanisms of HIV-1 infection and pathogenesis in the CNS that can be potentially served as therapeutic targets. The overall goal of this proposal is to determine the mechanisms of HIV-1 infection and pathogenesis in astrocytes. Recent studies from our laboratory demonstrated that HIV-1 infection of astrocytes is via a novel CD4-independent receptor 5C4R, and that direct binding of gp120 to 5C4R expressed on the cell surface induced 5C4R phosphorylation. Our studies also showed that astrocyte apoptosis induced by HIV-1 infection is a result of HIV-1 Nef expression in mitochondria. The underlying hypothesis for this proposal is that functional dysregulation of astrocytes by HIV-1 infection and interaction with HIV-1 viral protein gp120 and Nef results in, or contributes to HIV-1 neuropathogenesis. To test this hypothesis, we propose the following interrelated specific aims: 1) To characterize the newly-identified 5C4R receptor and its binding to gp120; 2) To determine mechanisms of 5C4R-mediated HIV-1 entry into astrocytes; 3) To characterize 5C4R-mediated signaling pathways in astrocytes upon gp120 binding; and 4) To determine mechanisms of Nef-induced apoptosis in 5C4R-mediated HIV-1 infection of astrocytes. Experimental approaches include the use of human fetal astrocyte cDNA expression cloning, 5C4R stable cell line, and an astroglial inducible cell line expressing HIV-1 Nef. These studies will yield insights that are fundamental to understanding the role of astrocytes in HIV-1 induced CNS pathology and will also elucidate the potential of HIV-1 Nef as targets for therapeutic intervention.